Device and process for isolating nucleic acids from cell suspension

ABSTRACT

A device and a process for isolating nucleic acids by lysing intact cells and removing nucleic acids emerging from the lysed cells by the following steps: 
     a) the cells are immobilized in a porous matrix, with the size of matrix voids being in the range of the type of cell to be lysed; 
     b) the cells are lysed; 
     c) the nucleic acids are fixated on the matrix surface, and subsequently 
     d) are eluted.

This application is a continuation of application Ser. No. 08/039,468,filed as PCT/EP91/0207 Oct. 24, 1991, now abandoned.

This invention is directed to a process for isolating nucleic acids bylysing intact cells and removing nucleic acids emerging from the lysedcells, and to a device for performing said process.

Purification of nucleic acids plays a central role in modern molecularbiology. Nucleic acids serve as starting materials for gene cloning andgenetic analyses in laboratory diagnostic research and in routine use.For example, analysis of genetic diseases of blood cells, virus andbacterial analytics from blood, urine, faeces, and secretions arecarried out on the basis of nucleic acids. Here, the analysis of wholeblood is of particular clinical importance.

Conventionally, nucleic acids are recovered from cells. For thispurpose, cells are digested, for instance, under severely denaturing andoptionally reducing conditions (Maniatis, T., Fritsch, E. F. & Sambrook,S., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring HarborUniversity Press, Cold Spring Harbor). Extensively used is digestion ofcells using detergents as denaturing agents and the use of specificenzymes to degrade protein structures and enzymes which cleave nucleicacids. Thus, for example, sodium dodecylsulfate (SDS) andethylenediamine tetraacetic acid (EDTA) are used as denaturing agents,and enzymes such as proteinase K. The result of such digestion proceduremostly is a highly viscous, gelatinous structure from which nucleicacids are isolated by phenol extraction. Here, the nucleic acids arepreserved with great length and are removed from the aqueous phasesubsequent to dialysis and precipitation. This digestion process is soaggressive that tissue fragments as well may be subjected to saidprocess. However, due to the labor-intensive technique involvingmultiple replacements of reaction vessels, such method is unfavorablefor large-scale sampling and routine preparations. While this process iscapable of being automatized, a commercial apparatus, however, currentlyperforms about 8 samples within four hours (Applied Biosystems A 371).Thus, the process is costly and is not suitable for passing samples inlarge series. Furthermore, it is disadvantageous that subsequentreactions such as enzymatic amplification are impaired due to the greatlength of the isolated nucleic acids. Moreover, the obtained solutionsare highly viscous and difficult to handle. In particular, DNA of greatlength tends to interfere since nucleic acids recovered by the processaccording to prior art must be sheared separately in order to be furtherprocessed.

Digestion of cells in alkaline medium in the presence of detergents istechnically simple but also results in nucleic acids of great length.

This crude preparation of nucleic acids is followed by subsequentreactions. These subsequent reactions require specific grade nucleicacids. Thus, the nucleic acid must be intact to the highest possibleextent and the yield of preparation must be high and reproducible. Thepreparation route must be simple and economic and must offer possibleautomatization. Nucleic acid preparation must be possible without therisk of cross contamination of other samples, particularly whereenzymatic amplification reactions such as Polymerase Chain Reaction(PCR) [Saiki, R., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi,R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988), Science 239,487-491] and Ligase Chain Reaction (LCR) (EP-A-88 311 741.8) areemployed. Therefore, it is desirable to obtain the nucleic acids withnot too great a chain length, to digest the cells as quantitative aspossible, and for what remains, to avoid the above-mentioneddisadvantages of digestion processes known in prior art.

Thus, the technical problem which this invention is based upon is toprovide an improved process for isolating nucleic acids from intactcells; in particular, the obtained nucleic acid should not exhibitexcessive chain length, should be isolable using a few steps, and shouldoffer the possibility of being subjected directly to the subsequentreactions required.

This technical problem is solved by a process for isolating nucleicacids by lysing intact cells and removing the nucleic acids emergingfrom the lysed cells, characterized in that

a) the cells are immobilized in a porous matrix, with the size of matrixvoids being in the range of the type of cell to be lysed;

b) the cells are lysed;

c) the nucleic acids are fixated on the matrix surface, and subsequently

d) are eluted.

Preferably, the matrix consists of porous inorganic or organic polymers,with the surface properties of the material, binding the matrixproviding for reversible or irreversible immobilization of cells at thesurface. Preferably, the void size of the material forming the matrix isfrom 1 to 50 μm. Particularly preferred are void sizes of from 5 to 15μm. This can be accomplished in a preferred embodiment of the processaccording to the invention wherein the particle size of the materialforming the matrix is from 10 to 50 μm, preferably from 15 to 25 μm.

The cells immobilized within the matrix are lysed preferably by physicalor chemical action where lysis either may be effected mechanically suchas by ultrasonic waves or by shear forces, osmotic shock, or chemicallyusing detergents or alkaline digestion.

In a particularly preferred embodiment, the surface of the materialforming the matrix has ion exchanging properties. Especially when usinganion exchangers the nucleic acid emerging from the lysed cell may bebound reversibly to the material forming the matrix to be eluted byadjusting to high ionic strengths subsequent to various washingoperations.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE illustrates a preferred embodiment of the device according tothe invention.

The procedure according to the invention results in preparation ofnucleic acids of high purity and permits to perform analyticsreproducible qualitatively and quantitatively, particularly incombination with enzymatic processes for the amplification of nucleicacids. It has turned out that mild digestion methods using detergents orphysical digestion methods such as heating a sample facilitatessubsequent applications. For instance, when using the process accordingto the invention, cellular DNA shorter in length (<200 kb) and totalnucleic acids, respectively, are obtained from the cell. The reasons forsuch limited nucleic acid cleavage are not completely known. However,the mild digestion methods appear to result in partial degradation ofthe nucleic acid.

For such reproducible procedure it is advantageous to use a matrixhaving ion exchanging properties so that liberated nucleic acids canadhere to this surface and are out of reach for degrading enzymes. Then,nucleic acids of nearly ideal length for subsequent reactions such asPCR are recovered.

For example, if white blood cells from mammals are to be digested,erythrocytes free of DNA must be removed first. To do this, variousprocesses are known such as, e.g., gradient centrifugation or extractionof cells using surface-coupled antibodies (anti-HLA magnetobeads). Ithas proven particularly advantageous to agglomerate the erythrocytesusing dextran and remove them without centrifugation. The supernatantcontains the other blood cells and less than 1/100 of the initialerythrocyte concentration. Then, the white blood cells are transferredfrom the suspension into the pores of the matrix using the processaccording to the invention. This is effected by pressure difference orcentrifugation forces. It has turned out that it is not sufficient toconcentrate the cells, for instance, by simple centrifugation, byforming a pellet, or by filtration through narrow-mesh membranes sincethen, cell digestion according to the invention is no longer effectedreliably. Cell formations thereby produced, being relatively dense andhaving direct cell-to-cell contact can be digested only insufficientlywhen using these mild non-ionic agents. Probably, merely those cells arelysed which are located at the surface of such a cell concentrate.

By the process according to the invention, it is ensured that suchpelletization does not occur. Here, the cells are concentrated by a typeof deep bed filtration within the matrix, i.e., are captured in a smallvolume element. This means that the cells are arranged within the matrixin virtually isolated fashion but do not lie on top of each other.According to the invention, this is achieved by using a porous matrix,for example, a particle bed, the interstices of which being in the rangeof the cell size. Then, the cells penetrate the matrix to a certaindepth.

In a preferred embodiment of the process according to the invention,silica gel particles are employed having a particle size of preferablyfrom 15 to 25 μm so that grain interspacings of from 1 to 50 μm areformed. The average size of erythrocytes is 2 μm, of lymphocytesapproximately 8 μm, and of monocytes about 15 μm.

In another preferred embodiment of the process according to theinvention, particles are used for immobilizing which are embedded in aninert network of membranes, preferably Teflon, the interstices againcorresponding approximately to the dimensions of the cells to be lysed.Thereby, it is ensured that the cells preferably are seized within thenetwork and do not--as for instance on a sterile filter--deposit denselypacked in the form of a filter cake.

Between the cells, narrow channels remain through which solutions can beinterchanged such as, for instance, serum for cell-lysing solution.Since all the cells are accessible for reagents, said mild lysingconditions may be used without the danger of losses in yield.

In a particularly preferred embodiment, the particles have ionexchanging properties as described, for example, in DE-PS 32 11 305. Forthe membrane-based embodiment, an ion exchanger on the basis of silicagel with particle sizes of between 15 and 25 μm and pore sizes of 1500 Åhas proven particularly useful. Due to direct spatial contact to all thelysing cells, it is ensured that the DNA, subsequent to lysis andpartial degradation, is directly fixed at the surface of the solid phaseand thus, is protected against further nuclease attack. In thiscondition, contaminants may be washed out readily, followed by elutingthe purified nucleic acid in a small volume. In this fashion,reproducible chain lengths of from 5 to 20 kb on average are obtained.Under the digestion conditions as described in the example, less than10% is shorter than 5 kb. This represents optimum length distributionfor subsequent enzymatic nucleic acid amplification. Likewisesurprisingly, it has been determined that the desired size distributionis achieved in leukocyte preparation using dextran while preparationover Ficoll in the gradient usually results in shorter fragments (500 to1000 bp).

It has turned out that the process according to the invention may beconducted in particularly favorable fashion by means of the deviceclaimed according to the invention and to be described in the following.The device according to the invention consists of a hollow body (1)wherein the matrix (4) accomodating the cells is arranged between twoporous units (2, 3). The pore size of the units 2, 3, preferablypolyethylene or glass frits, must be larger than the void size of thematerial forming the matrix 4. The units 2, 3 have a pore size of from50 to 100 μm, with the void size of the material forming the matrix 4being about from 1 to 50 μm. Matrix 4 is a net-like membrane having amultitude of pores of from 1 to 50 μm in size and in addition, ionexchanger particles. The cells are then immobilized within the freepores, with the liberated nucleic acids being adsorbed on the ionexchanger particles situated in said matrix.

A likewise preferred embodiment is represented by a device where thematerial forming the matrix 4 is a particulate organic or inorganicpolymer. Preferably, the material forming the matrix 4 is a silica gelhaving ion exchanging properties and a particle size of from 15 to 25μm.

The FIGURE illustrates a preferred embodiment of the device according tothe invention. For exmple, the hollow body 1 may be a commercial tubing(MoBiCol, MoBiTec, Gottingen or Millipore UFC3 OHW25). Between twonarrowly inserted units 2, 3, for example, polyethylene frits having apore size of from 50 to 100 μm, there is situated a membrane havingpores of from 5 to 10 μm in size and likewise containing silica gel(pore size 1500 Å, particle size 15 μm) having anion exchangingproperties. The membrane has a thickness of about 1 mm. The capacity ofthe ion exchanger material is about 15 μg of DNA. Of course, thecapacity for DNA may be increased by stacking corresponding membranesonto one another. With low mechanical lead, edge welding or cementingthe membrane is possible whereby the stabilizing effect of units 2, 3may be dropped so that the membrane seals the hollow body 4 without saidunits.

Likewise, it is possible to fill small columns with the described silicagel arranged between 2 polyethylene frits having a pore size of 35 μm.Preferably, the upper unit 2 is chosen with larger pores (70 μm). TheMoBiCol columns are charged with about 70 mg of ion exchanger gelcorresponding to a filling height of 3 mm. With respect to plasmid DNA,a capacity of 150 μg of DNA results.

EXAMPLE Cellular DNA Preparation from Blood

To 6 ml of citrate blood in a 12 ml PPN tube is homogeneously added 0.12g of dextran (MW 250,000) and incubated at room temperature for 60minutes. Dextran and erythrocytes form aggregates which sediment within45 minutes. The pale supernatant contains leukocytes in approximatelynatural composition [(1-5)×10⁶ leukocytes/ml, erythrocytes/leukocytesca. 2/1]. 0.5 ml of this supernatant is applied to the device and passedthrough the filter by centrifugation or pressure. The cells are retainedin the network. After passage of the serum, washing is effected using1×0.5 ml of washing solution (PBS 120 mM NaCl, 2.7 mM KCl, 10 mM KPi pH7.4) (phosphate buffered saline).

Thereafter, the cells are lysed using 125 μl of a 1% solution ofnon-ionic detergent (NP40; Tween 20). Impurities are washed out using0.5 ml of 750 mM KCl, 50 mM of MOPS pH 7.0, 15% ethanol. DNA is elutedusing either high saline buffer (1.2M KCl, 50 mM MOPS, pH 7.0) or alkalibuffer (200 mM KOH). The advantage of the alkali buffer consists in thatsubsequent to buffer addition for neutralization, direct enzymaticamplification may be effected, for example, without the necessity ofinitial nucleic acid precipitation. However, there is a risk of DNAdamage. In many cases, an aliquot of the high saline elution may be useddirectly if the required nucleic acid concentration is present.

What is claimed is:
 1. A process for isolating nucleic acids by lysingblood cells, substantially all of which are intact, and removing thenucleic acids emerging from the lysed cells, comprising the steps of:a)immobilizing the cells in a porous matrix having ion exchange propertiesand having voids each having a size sufficient to capture one of thecells; b) lysing the cells; c) fixing the emerging nucleic acids on thematrix surface by ion exchange, and subsequently; d) eluting the nucleicacids.
 2. The process according to claim 1, wherein the matrix has avoid size of from 1 to 50 μm.
 3. The process according to claim 2,wherein the void size is from 5 to 15 μm.
 4. The process according toclaim 1, wherein the porous matrix comprises particles each having aparticle size of 10 to 50 μm.
 5. The process according to claim 4,wherein the particle size is from 15 to 25 μm.
 6. The process accordingto claim 1, wherein the cell lysing is effected by ultrasonic waves,shear forces, detergents, enzymes, osmotic shock, or combinationsthereof.
 7. The process according to claim 1, wherein the nucleic acidsare eluted by a buffer or by alkaline elution.
 8. The process accordingto claim 1, wherein said matrix is washed between at least one of stepsa) and b), and b) and c), and c) and d).
 9. The process according toclaim 1, wherein the matrix is an inorganic or organic polymer.
 10. Theprocess according to claim 9, wherein the matrix has a void size of from1 to 50 μm.
 11. The process according to claim 10, wherein the void sizeis from 5 to 15 μm.
 12. In a process for isolating nucleic acids bylysing blood cells and removing the nucleic acids emerging from thelysed cells, the improvement comprising the steps of:a) before lysing,immobilizing the blood cells in a porous matrix having ion exchangeproperties and having voids each having a size sufficient to capture oneof the cells; b) lysing the captured cells; c) fixing the emergingnucleic acids on the matrix surface by ion exchange, and subsequently;d) eluting the nucleic acids.